MeCP2 binds to methylated DNA independently of phase separation and heterochromatin organisation.

Correlative evidence has suggested that the methyl-CpG-binding protein MeCP2 contributes to the formation of heterochromatin condensates via liquid-liquid phase separation. This interpretation has been reinforced by the observation that heterochromatin, DNA methylation and MeCP2 co-localise within prominent foci in mouse cells. The findings presented here revise this view. MeCP2 localisation is independent of heterochromatin as MeCP2 foci persist even when heterochromatin organisation is disrupted. Additionally, MeCP2 foci fail to show hallmarks of phase separation in live cells. Importantly, we find that mouse cellular models are highly atypical as MeCP2 distribution is diffuse in most mammalian species, including humans. Notably, MeCP2 foci are absent in Mus spretus which is a mouse subspecies lacking methylated satellite DNA repeats. We conclude that MeCP2 has no intrinsic tendency to form condensates and its localisation is independent of heterochromatin. Instead, the distribution of MeCP2 in the nucleus is primarily determined by global DNA methylation patterns.

The holocentricity in the dioecious nutmeg (Myristica fragrans) is not based on major satellite repeats.

Holocentric species are characterized by the presence of centromeres throughout the length of the chromosomes. We confirmed the holocentricity of the dioecious, small chromosome-size species Myristica fragrans based on the chromosome-wide distribution of the centromere-specific protein KNL1, α-tubulin fibers, and the cell cycle-dependent histone H3 serine 28 phosphorylation (H3S28ph) mark. Each holocentromere is likely composed of, on average, ten centromere units, but none of the identified and in situ hybridized high-copy satellite repeats is centromere-specific. No sex-specific major repeats are present in the high-copy repeat composition of male or female plants, or a significant difference in genome size was detected. Therefore, it is unlikely that M. fragrans possesses heteromorphic sex chromosomes.

Novel Concept of Alpha Satellite Cascading Higher-Order Repeats (HORs) and Precise Identification of 15mer and 20mer Cascading HORs in Complete T2T-CHM13 Assembly of Human Chromosome 15.

Unraveling the intricate centromere structure of human chromosomes holds profound implications, illuminating fundamental genetic mechanisms and potentially advancing our comprehension of genetic disorders and therapeutic interventions. This study rigorously identified and structurally analyzed alpha satellite higher-order repeats (HORs) within the centromere of human chromosome 15 in the complete T2T-CHM13 assembly using the high-precision GRM2023 algorithm. The most extensive alpha satellite HOR array in chromosome 15 reveals a novel cascading HOR, housing 429 15mer HOR copies, containing 4-, 7- and 11-monomer subfragments. Within each row of cascading HORs, all alpha satellite monomers are of distinct types, as in regular Willard's HORs. However, different HOR copies within the same cascading 15mer HOR contain more than one monomer of the same type. Each canonical 15mer HOR copy comprises 15 monomers belonging to only 9 different monomer types. Notably, 65% of the 429 15mer cascading HOR copies exhibit canonical structures, while 35% display variant configurations. Identified as the second most extensive alpha satellite HOR, another novel cascading HOR within human chromosome 15 encompasses 164 20mer HOR copies, each featuring two subfragments. Moreover, a distinct pattern emerges as interspersed 25mer/26mer structures differing from regular Willard's HORs and giving rise to a 34-monomer subfragment. Only a minor 18mer HOR array of 12 HOR copies is of the regular Willard's type. These revelations highlight the complexity within the chromosome 15 centromeric region, accentuating deviations from anticipated highly regular patterns and hinting at profound information encoding and functional potential within the human centromere.

Heterochromatin Is Not the Only Place for satDNAs: The High Diversity of satDNAs in the Euchromatin of the Beetle Chrysolina americana (Coleoptera, Chrysomelidae).

The satellitome of the beetle Chrysolina americana Linneo, 1758 has been characterized through chromosomal analysis, genomic sequencing, and bioinformatics tools. C-banding reveals the presence of constitutive heterochromatin blocks enriched in A+T content, primarily located in pericentromeric regions. Furthermore, a comprehensive satellitome analysis unveils the extensive diversity of satellite DNA families within the genome of C. americana. Using fluorescence in situ hybridization techniques and the innovative CHRISMAPP approach, we precisely map the localization of satDNA families on assembled chromosomes, providing insights into their organization and distribution patterns. Among the 165 identified satDNA families, only three of them exhibit a remarkable amplification and accumulation, forming large blocks predominantly in pericentromeric regions. In contrast, the remaining, less abundant satDNA families are dispersed throughout euchromatic regions, challenging the traditional association of satDNA with heterochromatin. Overall, our findings underscore the complexity of repetitive DNA elements in the genome of C. americana and emphasize the need for further exploration to elucidate their functional significance and evolutionary implications.

Cytogenomic Characterization of Transposable Elements and Satellite DNA in Passiflora L. Species.

The species Passiflora alata, P. cincinnata, and P. edulis have great economic value due to the use of their fruits for human consumption. In this study, we compared the repetitive genome fractions of these three species. The compositions of the repetitive DNA of these three species' genomes were analyzed using clustering and identification of the repetitive sequences with RepeatExplorer. It was found that repetitive DNA content represents 74.70%, 66.86%, and 62.24% of the genome of P. alata, P. edulis, and P. cincinnata, respectively. LTR Ty3/Gypsy retrotransposons represent the highest genome proportions in P. alata and P. edulis, while Ty1/Copia comprises the largest proportion of P. cincinnata genome. Chromosomal mapping by Fluorescent In Situ Hybridization (FISH) showed that LTR retrotransposons have a dispersed distribution along chromosomes. The subtelomeric region of chromosomes is where 145 bp satellite DNA is located, suggesting that these elements may play important roles in genome structure and organization in these species. In this work, we obtained the first global characterization of the composition of repetitive DNA in Passiflora, showing that an increase in genome size is related to an increase in repetitive DNA, which represents an important evolutionary route for these species.

Cytogenetic Analysis of Satellitome of Madagascar Leaf-Tailed Geckos.

Satellite DNA (satDNA) consists of sequences of DNA that form tandem repetitions across the genome, and it is notorious for its diversity and fast evolutionary rate. Despite its importance, satDNA has been only sporadically studied in reptile lineages. Here, we sequenced genomic DNA and PCR-amplified microdissected W chromosomes on the Illumina platform in order to characterize the monomers of satDNA from the Henkel's leaf-tailed gecko U. henkeli and to compare their topology by in situ hybridization in the karyotypes of the closely related Günther's flat-tail gecko U. guentheri and gold dust day gecko P. laticauda. We identified seventeen different satDNAs; twelve of them seem to accumulate in centromeres, telomeres and/or the W chromosome. Notably, centromeric and telomeric regions seem to share similar types of satDNAs, and we found two that seem to accumulate at both edges of all chromosomes in all three species. We speculate that the long-term stability of all-acrocentric karyotypes in geckos might be explained from the presence of specific satDNAs at the centromeric regions that are strong meiotic drivers, a hypothesis that should be further tested.

The variation and evolution of complete human centromeres.

Human centromeres have been traditionally very difficult to sequence and assemble owing to their repetitive nature and large size1. As a result, patterns of human centromeric variation and models for their evolution and function remain incomplete, despite centromeres being among the most rapidly mutating regions2,3. Here, using long-read sequencing, we completely sequenced and assembled all centromeres from a second human genome and compared it to the finished reference genome4,5. We find that the two sets of centromeres show at least a 4.1-fold increase in single-nucleotide variation when compared with their unique flanks and vary up to 3-fold in size. Moreover, we find that 45.8% of centromeric sequence cannot be reliably aligned using standard methods owing to the emergence of new α-satellite higher-order repeats (HORs). DNA methylation and CENP-A chromatin immunoprecipitation experiments show that 26% of the centromeres differ in their kinetochore position by >500 kb. To understand evolutionary change, we selected six chromosomes and sequenced and assembled 31 orthologous centromeres from the common chimpanzee, orangutan and macaque genomes. Comparative analyses reveal a nearly complete turnover of α-satellite HORs, with characteristic idiosyncratic changes in α-satellite HORs for each species. Phylogenetic reconstruction of human haplotypes supports limited to no recombination between the short (p) and long (q) arms across centromeres and reveals that novel α-satellite HORs share a monophyletic origin, providing a strategy to estimate the rate of saltatory amplification and mutation of human centromeric DNA.

Amplification of different satellite-DNAs in prostate cancer.

In various solid tumors and corresponding cell lines, prior research has identified acquired copy number variations (CNVs) encompassing centromeric satellite-DNA sequences. This observation emerged from the application of centromeric probes (satellite-DNA) as controls in molecular cytogenetic investigations and diagnostics, although these accounts were largely anecdotal. In this study, we conducted a systematic screening for satellite-DNA sequence amplification in 31 prostate cancer (PCa) samples, a prevalent malignancy in men characterized by discernible molecular cytogenetic aberrations. Notably, PCa-typical genetic aberrations, such as TMPRSS2-ERG gene rearrangements and PTEN deletion, were identified in 12 and 6 out of the 31 PCa samples, respectively. Overall, PCa exhibited genomic instability marked by chromosomal gain or loss of signals across nearly all tested satellite-DNA regions, with particular emphasis on the Y-chromosome (18/31 cases). Remarkably, 5/12 PCa samples representing more advanced metastatic cancer displayed amplification of one or two satellite DNA stretches each, being detectable as blocks analogous to homogenously staining regions. Notably, these stretches included α-satellite DNA derived from chromosomes 2, 3, 4, 15, and 20, as well as satellite-III DNAs (D1Z1 and DYZ1). These findings align with recent discoveries indicating that α-satellite DNAs are expressed as long-non-coding RNAs in advanced cancer, particularly in the context of PCa.

Divergence of 10 satellite repeats in Artemisia (Asteraceae: Anthemideae) based on sequential fluorescence in situ hybridization analysis: evidence for species identification and evolution.

Artemisia is a large genus encompassing about 400 diverse species, many of which have considerable medicinal and ecological value. However, complex morphological information and variation in ploidy level and nuclear DNA content have presented challenges for evolution studies of this genus. Consequently, taxonomic inconsistencies within the genus persist, hindering the utilization of such large plant resources. Researchers have utilized satellite DNAs to aid in chromosome identification, species classification, and evolutionary studies due to their significant sequence and copy number variation between species and close relatives. In the present study, the RepeatExplorer2 pipeline was utilized to identify 10 satellite DNAs from three species (Artemisia annua, Artemisia vulgaris, Artemisia viridisquama), and fluorescence in situ hybridization confirmed their distribution on chromosomes in 24 species, including 19 Artemisia species with 5 outgroup species from Ajania and Chrysanthemum. Signals of satellite DNAs exhibited substantial differences between species. We obtained one genus-specific satellite from the sequences. Additionally, molecular cytogenetic maps were constructed for Artemisia vulgaris, Artemisia leucophylla, and Artemisia viridisquama. One species (Artemisia verbenacea) showed a FISH distribution pattern suggestive of an allotriploid origin. Heteromorphic FISH signals between homologous chromosomes in Artemisia plants were observed at a high level. Additionally, the relative relationships between species were discussed by comparing ideograms. The results of the present study provide new insights into the accurate identification and taxonomy of the Artemisia genus using molecular cytological methods.

Variants of a major DNA satellite discriminate parental subgenomes in a hybrid parthenogenetic lizard Darevskia unisexualis (Darevsky, 1966).

Hybrid parthenogenetic animals are an exceptionally interesting model for studying the mechanisms and evolution of sexual and asexual reproduction. A diploid parthenogenetic lizard Darevskia unisexualis is a result of an ancestral cross between a maternal species Darevskia raddei nairensis and a paternal species Darevskia valentini and presents a unique opportunity for a cytogenetic and computational analysis of a hybrid karyotype. Our previous results demonstrated a significant divergence between the pericentromeric DNA sequences of the parental Darevskia species; however, an in-depth comparative study of their pericentromeres is still lacking. Here, using target sequencing of microdissected pericentromeric regions, we reveal and compare the repertoires of the pericentromeric tandem repeats of the parental Darevskia lizards. We found species-specific sequences of the major pericentromeric tandem repeat CLsat, which allowed computational prediction and experimental validation of fluorescent DNA probes discriminating parental chromosomes within the hybrid karyotype of D. unisexualis. Moreover, we have implemented a generalizable computational method, based on the optimization of the Levenshtein distance between tandem repeat monomers, for finding species-specific fluorescent probes for pericentromere staining. In total, we anticipate that our comparative analysis of Darevskia pericentromeric repeats, the species-specific fluorescent probes that we found and the pipeline that we developed will form a basis for the future detailed cytogenomic studies of a wide range of natural and laboratory hybrids.

Satellite DNAs, heterochromatin, and sex chromosomes of the wattled jacana (Charadriiformes; Jacanidae): a species with highly rearranged karyotype.

Charadriiformes, which comprises shorebirds and their relatives, is one of the most diverse avian orders, with over 390 species showing a wide range of karyotypes. Here, we isolated and characterized the whole collection of satellite DNAs (satDNAs) at both molecular and cytogenetic levels of one of its representative species, named the wattled jacana (Jacana jacana), a species that contains a typical ZZ/ZW sex chromosome system and a highly rearranged karyotype. In addition, we also investigate the in situ location of telomeric and microsatellite repeats. A small catalog of 11 satDNAs was identified that typically accumulated on microchromosomes and on the W chromosome. The latter also showed a significant accumulation of telomeric signals, being (GA)10 the only microsatellite with positive hybridization signals among all the 16 tested ones. These current findings contribute to our understanding of the genomic organization of repetitive DNAs in a bird species with high degree of chromosomal reorganization contrary to the majority of bird species that have stable karyotypes.

Differential amplification of the subtelomeric satellite DNA JcSAT1 in the genus Jatropha L. (Euphorbiaceae).

Satellite DNAs (satDNAs) are highly repetitive sequences that occur in virtually all eukaryotic genomes and can undergo rapid copy number and nucleotide sequence variation among relatives. After chromosomal mapping of the satDNA JcSAT1, it was found a large accumulation at subtelomeres of Jatropha curcas (subgenus Curcas), but an absence of these monomers in J. integerrima (subgenus Jatropha). This fact suggests a dynamic scenario for this satellite repeat in Jatropha genomes. Here, we used a multitasking approach (sequence analysis, DNA blotting and chromosomal mapping) to investigate the molecular organization and chromosomal abundance and distribution of JcSAT1 in a broader group of species from the subgenus Jatropha (J. gossypiifolia, J. mollissima, J. podagrica, and J. multifida) in addition to J. curcas, with the aiming of understanding the evolution of this satDNA. Based on the analysis of BAC clone sequences of J. curcas, a large array (~ 30 kb) of 80 homogeneous monomers of JcSAT1 was identified in BAC 23J11. The monomer size was conserved (~ 358 bp) and contained a telomeric motif at the 5' end. PCR amplification coupled with a Southern blot revealed the presence of JcSAT1-like sequences in all species examined. However, a large set of genome copies was identified only in J. curcas, where a ladder-like pattern with multimers of different sizes was observed. In situ hybridization of BAC 23J11 confirmed the subtelomeric pattern for J. curcas, but showed no signals on chromosomes of species from the subgenus Jatropha. Our data indicate that JcSAT1 is a highly homogeneous satDNA that originated from a region near the telomeres and spread throughout the chromosomal subtermini, possibly due to frequent ectopic recombination between these regions. The abundance of JcSAT1 in the genome of J. curcas suggests that an amplification event occurred either at the base of the subgenus Curcas or at least in this species, although the repeat is shared by all species of the genus studied so far.

Satellite double-stranded RNA induces mesenchymal transition in pancreatic cancer by regulating alternative splicing.

Human satellite II (HSATII), composed of tandem repeats in pericentromeric regions, is aberrantly transcribed in epithelial cancers, particularly pancreatic cancer. Dysregulation of repetitive elements in cancer tissues can facilitate incidental dsRNA formation; however, it remains controversial whether dsRNAs play tumor-promoting or tumor-suppressing roles during cancer progression. Therefore, we focused on the double-stranded formation of HSATII RNA and explored its molecular function. The overexpression of double-stranded HSATII (dsHSATII) RNA promoted mesenchymal-like morphological changes and enhanced the invasiveness of pancreatic cancer cells. We identified an RNA-binding protein, spermatid perinuclear RNA-binding protein (STRBP), which preferentially binds to dsHSATII RNA rather than single-stranded HSATII RNA. The mesenchymal transition of dsHSATII-expressing cells was rescued by STRBP overexpression. Mechanistically, STRBP is involved in the alternative splicing of genes associated with epithelial-mesenchymal transition (EMT). We also confirmed that isoform switching of CLSTN1, driven by dsHSATII overexpression or STRBP depletion, induced EMT-like morphological changes. These findings reveal a novel tumor-promoting function of dsHSATII RNA, inducing EMT-like changes and cell invasiveness, thus enhancing our understanding of the biological significance of aberrant expression of satellite arrays in malignant tumors.

Evolution of ancient satellite DNAs in extant alligators and caimans (Crocodylia, Reptilia).

BACKGROUND: Crocodilians are one of the oldest extant vertebrate lineages, exhibiting a combination of evolutionary success and morphological resilience that has persisted throughout the history of life on Earth. This ability to endure over such a long geological time span is of great evolutionary importance. Here, we have utilized the combination of genomic and chromosomal data to identify and compare the full catalogs of satellite DNA families (satDNAs, i.e., the satellitomes) of 5 out of the 8 extant Alligatoridae species. As crocodilian genomes reveal ancestral patterns of evolution, by employing this multispecies data collection, we can investigate and assess how satDNA families evolve over time. RESULTS: Alligators and caimans displayed a small number of satDNA families, ranging from 3 to 13 satDNAs in A. sinensis and C. latirostris, respectively. Together with little variation both within and between species it highlighted long-term conservation of satDNA elements throughout evolution. Furthermore, we traced the origin of the ancestral forms of all satDNAs belonging to the common ancestor of Caimaninae and Alligatorinae. Fluorescence in situ experiments showed distinct hybridization patterns for identical orthologous satDNAs, indicating their dynamic genomic placement. CONCLUSIONS: Alligators and caimans possess one of the smallest satDNA libraries ever reported, comprising only four sets of satDNAs that are shared by all species. Besides, our findings indicated limited intraspecific variation in satellite DNA, suggesting that the majority of new satellite sequences likely evolved from pre-existing ones.

Satellitome Analysis in the Southern Lapwing (Vanellus chilensis) Genome: Implications for SatDNA Evolution in Charadriiform Birds.

Vanellus (Charadriidae; Charadriiformes) comprises around 20 species commonly referred to as lapwings. In this study, by integrating cytogenetic and genomic approaches, we assessed the satellite DNA (satDNA) composition of one typical species, Vanellus chilensis, with a highly conserved karyotype. We additionally underlined its role in the evolution, structure, and differentiation process of the present ZW sex chromosome system. Seven distinct satellite DNA families were identified within its genome, accumulating on the centromeres, microchromosomes, and the W chromosome. However, these identified satellite DNA families were not found in two other Charadriiformes members, namely Jacana jacana and Calidris canutus. The hybridization of microsatellite sequences revealed the presence of a few repetitive sequences in V. chilensis, with only two out of sixteen displaying positive hybridization signals. Overall, our results contribute to understanding the genomic organization and satDNA evolution in Charadriiform birds.

The implications of satellite DNA instability on cellular function and evolution.

Abundant tandemly repeated satellite DNA is present in most eukaryotic genomes. Previous limitations including a pervasive view that it was uninteresting junk DNA, combined with challenges in studying it, are starting to dissolve - and recent studies have found important functions for satellite DNAs. The observed rapid evolution and implied instability of satellite DNA now has important significance for their functions and maintenance within the genome. In this review, we discuss the processes that lead to satellite DNA copy number instability, and the importance of mechanisms to manage the potential negative effects of instability. Satellite DNA is vulnerable to challenges during replication and repair, since it forms difficult-to-process secondary structures and its homology within tandem arrays can result in various types of recombination. Satellite DNA instability may be managed by DNA or chromatin-binding proteins ensuring proper nuclear localization and repair, or by proteins that process aberrant structures that satellite DNAs tend to form. We also discuss the pattern of satellite DNA mutations from recent mutation accumulation (MA) studies that have tracked changes in satellite DNA for up to 1000 generations with minimal selection. Finally, we highlight examples of satellite evolution from studies that have characterized satellites across millions of years of Drosophila fruit fly evolution, and discuss possible ways that selection might act on the satellite DNA composition.

Dynamic interplay between human alpha-satellite DNA structure and centromere functions.

Maintenance of genome stability relies on functional centromeres for correct chromosome segregation and faithful inheritance of the genetic information. The human centromere is the primary constriction within mitotic chromosomes made up of repetitive alpha-satellite DNA hierarchically organized in megabase-long arrays of near-identical higher order repeats (HORs). Centromeres are epigenetically specified by the presence of the centromere-specific histone H3 variant, CENP-A, which enables the assembly of the kinetochore for microtubule attachment. Notably, centromeric DNA is faithfully inherited as intact haplotypes from the parents to the offspring without intervening recombination, yet, outside of meiosis, centromeres are akin to common fragile sites (CFSs), manifesting crossing-overs and ongoing sequence instability. Consequences of DNA changes within the centromere are just starting to emerge, with unclear effects on intra- and inter-generational inheritance driven by centromere's essential role in kinetochore assembly. Here, we review evidence of meiotic selection operating to mitigate centromere drive, as well as recent reports on centromere damage, recombination and repair during the mitotic cell division. We propose an antagonistic pleiotropy interpretation to reconcile centromere DNA instability as both driver of aneuploidy that underlies degenerative diseases, while also potentially necessary for the maintenance of homogenized HORs for centromere function. We attempt to provide a framework for this conceptual leap taking into consideration the structural interface of centromere-kinetochore interaction and present case scenarios for its malfunctioning. Finally, we offer an integrated working model to connect DNA instability, chromatin, and structural changes with functional consequences on chromosome integrity.

Defining a core configuration for human centromeres during mitosis.

The centromere components cohesin, CENP-A, and centromeric DNA are essential for biorientation of sister chromatids on the mitotic spindle and accurate sister chromatid segregation. Insight into the 3D organization of centromere components would help resolve how centromeres function on the mitotic spindle. We use ChIP-seq and super-resolution microscopy with single particle averaging to examine the geometry of essential centromeric components on human chromosomes. Both modalities suggest cohesin is enriched at pericentromeric DNA. CENP-A localizes to a subset of the α-satellite DNA, with clusters separated by ~562 nm and a perpendicular intervening ~190 nM wide axis of cohesin in metaphase chromosomes. Differently sized α-satellite arrays achieve a similar core structure. Here we present a working model for a common core configuration of essential centromeric components that includes CENP-A nucleosomes, α-satellite DNA and pericentromeric cohesion. This configuration helps reconcile how centromeres function and serves as a foundation to add components of the chromosome segregation machinery.

Alpha Satellite DNA in Targeted Drug Therapy for Prostate Cancer.

Prostate cancer is the most common solid cancer in men and, despite the development of many new therapies, metastatic castration-resistant prostate cancer still remains a deadly disease. Therefore, novel concepts for the treatment of metastatic prostate cancer are needed. In our opinion, the role of the non-coding part of the genome, satellite DNA in particular, has been underestimated in relation to diseases such as cancer. Here, we hypothesise that this part of the genome should be considered as a potential target for the development of new drugs. Specifically, we propose a novel concept directed at the possible treatment of metastatic prostate cancer that is mostly based on epigenetics. Namely, metastatic prostate cancer is characterized by the strongly induced transcription of alpha satellite DNA located in pericentromeric heterochromatin and, according to our hypothesis, the stable controlled transcription of satellite DNA might be important in terms of the control of disease development. This can be primarily achieved through the epigenetic regulation of pericentromeric heterochromatin by using specific enzymes as well as their activators/inhibitors that could act as potential anti-prostate cancer drugs. We believe that our concept is innovative and should be considered in the potential treatment of prostate cancer in combination with other more conventional therapies.